Herpesviruses are ubiquitous and are responsible for many important human diseases. Perhaps the least understood steps in their replication are the cytoplasmic events of budding and egress, which we are investigating with the most thoroughly characterized herpesvirus, HSV-1. The UL11 protein has been the primary focus of our studies because it has homologs in all other herpesviruses, and whenever these are absent, budding is impaired and capsids accumulate in the cytoplasm. Moreover, UL11 localizes at the TON (the site of budding), even when express in the absence of other viral proteins. However, it also trafficks to the plasma membrane and can move in and out of lipid-raft domains, raising the possibility that its function resides outside the TGN. A search for ULU-binding partners revealed two unanticipated proteins, UL16 and UL21, which have homologs in all herpesviruses, but their specific functions and mechanisms of action are also unknown. These two proteins are found in the nucleus, cytoplasm, and mature virions, but their sites of accumulation change with time. In particular, UL16 (and probably UL21) is found in the nucleus when its expression begins, but at later times, it is found primarily on cytoplasmic membranes. However, the most novel observation regarding UL16 and UL21 is that they associate with capsids in a transient manner. Specifically, they are found only on capsids isolated from the cytoplasm but not those from the nucleus or virions. We hypothesize that UL16 and UL21 are involved in transporting capsids to the TGN, where they dock with UL11 and then release their cargo. In a parallel search for components of the budding machinery, we have examined virions for the presence of proteins that are modified with ubiquitin. This approach was based on our previous discovery that Ub is required for retrovirus budding. Several lib-modified proteins were found, some of which are tightly associated with capsids. To pursue these clues to the mechanism of HSV-1 assembly and budding, we have devised a comprehensive set of experiments that take advantage of the combined expertise of four experienced investigators: John Wills (virus budding), Richard Courtney (HSV-1 proteins), Vincent Chau (ubiquitin-mediated events), and John Semmes (mass spectrometry). Together, we propose to investigate: 1) the function of UL11 in HSV-1 budding, 2) the functions of UL16 and UL21, and 3) the identities and importance of Ub-modified proteins associated with HSV-1 capsids.